Activation of the trigeminovascular pathway: functional relationship with CSD, dural mast cells,and parasympathetic innervation of the dura. Migraine is a unilateral throbbing headache usually heralded by visual aura, as well as other premonitory symptoms, beginning several minutes earlier. Large bodies of evidence have suggested that visual aura is driven by cortical spreading depression (CSD), whereas migraine headache by activation of meningeal nociceptors. In this grant proposal, we aim at gaining insights into novel mechanisms by which CSD may lead to the activation of meningeal nociceptors. Four working hypotheses will be tested. Under hypothesis 1, we will test whether meningeal nociceptors can be activated and sensitized by CSD. Under hypothesis 2, we will test whether activation and degranulation of meningeal mast cells mediate CSD-induced activation/sensitization of meningeal nociceptors. Under hypothesis 3, we will test whether the parasympathetic nervous system mediates sustained activation/sensitization of meningeal nociceptors induced by CSD. Under hypothesis 4, we will test whether CSD can activate and sensitize central trigeminovascular neurons in laminae I and V of the medullary dorsal horn. We will employ single-unit recording to study A5 and C-fiber meningeal nociceptors and central trigeminovascular neurons, and histological techniques to study morphological and biochemical changes in the dura. In Specific Aim 1a, meningeal nociceptors will be studied for their ongoing activity and physiological response properties before and after experimental induction of CSD. In Specific Aim 1/>, we will test the effect of CSD on dural expression of pERK, a surrogate marker for nociceptive activation. In Specific aim 2, meningeal nociceptors will be studied electrophysiologically before and after induction of CSD in rats that were pretreated (prior to CSD) for depletion of mast cells (using compound 48/80) or inhibition of mast cell degranulation (using sodium cromoglycate). In Specific aim 3, meningeal nociceptors will be studied electrophysiologically before and after induction of CSD in rats devoid of parasympathetic innervation of the dura (following ablation of the sphenopalatine ganglion). In Specific Aim 4, trigeminovascular neurons in laminae I and V of the dorsal horn will be studied electrophysiologically before and after CSD. Prior to this grant proposal, we have studied the effects of local application of inflammatory mediators to the dura and mast cell degranulation on neuronal activation and sensitization in the trigeminal ganglion and medullary dorsal horn. The studies proposed herein will are a natural 'next step'in our quest to understand better how meningeal nociceptors are activates during migraine.